中文摘要：

探讨冷藏是否导致新鲜冰冻血浆（fresh frozen plasma，FFP）中的转化生长因子-β（transform growth factor-β，TGF-β）发生变化并降低其对内皮细胞迁移的诱导能力及其分子机制．为证实冷藏可能导致FFP中TGF-β的水平升高进而影响其功能，首先采用ELISA法分析当天解冻FFP（FFPDayO）和解冻后4℃冷藏1～5天FFP中TGF—β含量，迁移实验及Western blot分析比较FFP（DayO）和FFP（Day5）处理人肺微血管内皮细胞（human pulmonary microvascular endothelial cells，HPMECs）后的细胞迁移率及TGF-β信号通路Smad2和Smad3磷酸化，进一步采用ALK5-siRNA和ALK5特异性抑制剂阻断细胞TGF-βI型受体ALK5的活性，分析下调TGF-β／Smad2／3信号传导后的内皮细胞迁移率．结果显示：随着冷藏时间的延长，FFP中TGF-β1水平逐渐增加，其增加率为244．31ned（L·d）（P〈0．05）；与FFP（Day 0）相比，FFP（Day 5）诱导内皮细胞HPMECs的TGF-β信号通路Smad2／3磷酸化显著增加（P〈0．05）；不管是FFP（Day0）还是FFP（Day5）体外均能诱导内皮细胞迁移，与FFP（Day 0）相比，FFP（Day5）诱导细胞迁移能力显著降低（P〈0．05）；阻断TGF-βI型受体ALK5的活性、下调Smad3信号传导可恢复FFP（Day5）诱导细胞迁移能力．上述结果表明，FFP能显著诱导内皮细胞迁移，4℃冷藏增加FFP中TGF-β1水平并增加内皮细胞TGF-β1信号传导，进而降低FFP诱导内皮细胞迁移．提示，FFP复苏疗效的提高可能与增加内皮细胞迁移有关，冷藏导致内皮细胞迁移降低进而降低FFP复苏疗效．

英文摘要：

To determine whether TGF-βs and the effect on endothelial cell migration were altered during fresh frozen plasma （FFP） refrigeration, ELISA assay was carried out to quantify TGF-βs protein levels in FFP stored at 4 ℃ for up to 5 days. Human pulmonary microvascular endothelial cells （HPMECs） were treated with various concentrations of Day 0 and Day 5 FFPs or 10% Lactated Ringer＇s （LR） and were subjected to migration assay or Western blot analysis. ALK5 siRNA or ALK5 inhibitor were used to block FFP-induced Smad3 signaling in EC cells. It was found that TGF-β1 protein levels increased in a time-dependent fashion at a rate of 244.31 ng/（L ·d） （P 〈 0.05） and greater activation of its downstream mediators Smad2/3 during storage of FFP （P 〈 0.05）. Both Day 0 FFP and Day 5 FFP stimulated EC migration in vitro; however, the effect of Day 5 FFP was significantly reduced. Inhibition of TGF-β type I receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of Day 5 FFP on EC migration to a comparable level as Day 0 FFP. These data suggest that the increased TGF-β levels during FFP storage contributes to the deterioration of stored FFP＇s effects on EC migration. A novel molecular mechanism contributing to the reduced efficacy of stored FFP was identified.